Effect of feed trace elements on eggs of five strains of laying hens and their health risk assessment

Abstract Background Trace elements are essential for human nutrition, and their deficiencies or excesses are strongly associated with several diseases, such as cardiovascular ones. Objectives The current cross‐sectional study investigated the concentration of essential trace elements (copper, non‐metal selenium, iron, zinc, cobalt and manganese) in eggs and diets of five strains of laying hens. Methods The yolk and albumen were analysed separately, and wet preparation was carried out following inductively coupled plasma–optical emission spectrometry detection. The target hazard quotients (THQs) for the non‐carcinogenic disease were calculated by the United States environmental protection agency (USEPA) method. Results The highest concentrations of selenium, zinc and manganese were found in egg yolks of native hens (0.76, 44.22 and 6.52 mg/kg, respectively). The highest amounts of copper and cobalt were recorded in the egg yolk of Lohman (2.07 and 0.023 mg/kg, respectively). On the other hand, the egg yolk of Bovans contained the highest amount of iron (57.46 mg/kg). Conclusion Overall, the potential health risks were minimal, and the consumption of eggs was generally safe.

Furthermore, some diseases like cancer, cardiovascular diseases and diabetes have also been associated with trace elements (Fraga, 2005;Yang et al., 2022).
Insufficient or excessive amounts of trace elements are both harmful. Moreover, essential metals can be accumulated in organs, if excessive quantities enter the body. This does not only happen in humans but also avians. Avians also need trace elements like Cu, non-metal Se, Zn, Fe, Co, Mn, chromium (Cr), lithium (Li), molybdenum (Mo) and nickel (Ni) for their normal development (Noetzold et al., 2022). Polluted feed, water, environmental factors like outdoor and indoor air, veterinary medicines and mineral supplements may transfer excessive amounts of trace elements into eggs (Dobrzański et al., 2007;Vincevica-Gaile et al., 2013).
On the other hand, the egg is a high-quality foodstuff, and in comparison with other protein sources, it is of lower cost. The egg consists of a shell, membrane shells, albumen and yolk (Yao et al., 2014). The egg is consisted of shell, membrane shells, albumen and yolk (Yao et al., 2014). It is also regarded as a vital source of non-metal Se in countries like Japan, Portugal and Greece (Pinto et al., 2016). At the same time, it contains a variety of organic compounds such as proteins and lipids, with the ability to bind trace elements ions like ovalbumin (an egg white protein), which chelates with Zn, Cu and Mn, or egg yolk lipids, which chelate with Cr, Zn, Ni (Vincevica-Gaile et al., 2013).
The chicken strain could influence egg composition, size, shell thickness and weight (Pinto et al., 2016;Scott & Silversides, 2000;Yao et al., 2014). Recently, many studies focused on the productive performance parameters (feed intake, egg production, shell thickness, egg weight, shell weight etc.) of hens among different strains (Ershad, 2005;Scheideler et al., 1998;Scott & Silversides, 2000;Singh et al., 2009) and diet or concentration of trace elements of eggs (Abdulkhaliq et al., 2012;Ali et al., 2016;Bologa et al., 2014;Demirulus, 2013;Korish & Attia, 2020;Uluozlu et al., 2009;Vincevica-Gaile et al., 2013;Waegeneers et al., 2009). However, there is no study focusing on the impact of laying hens strains on the concentration of micronutrients in the egg. Thus, the current study was conducted to examine: (1) the concentration of essential trace elements (Cu, none-metal Se, Fe, Zn, Co and Mn) of eggs and diets in five strains, including Hy-Line W36, Shaver White, Lohman LSL-Lite, native hens (Gallus gallus domesticus) and Bovans White; (2) the association among the strain, the diet and the concentration of essential metals in the eggs; (3) the none-carcinogenic health risk assessment via the consumption of egg.

Sample collection
Five strains in cages system farms offering different commercial feeds and then with distilled water (Pinto et al., 2016;Vincevica-Gaile et al., 2013).

Egg and feed analyses
Egg samples were kept at 5 • C until analysis. The egg white, egg yolk and feed samples were prepared by a method previously described by

Apparatus and trace element detection
The toxic trace elements: Cu, none-metal Se, Mn, Zn, Fe and Co in feeds and eggs were determined using inductively coupled plasma-optical emission spectrometry (ICP-OES) (SPECTRO-ARCOS, GERMANY).
The operation parameters for detection were 1 L/min nebulizer flow and 2.5 mm fixed plasma torch (Table 1). Multi-element standard solutions (Carlo Erba reagents) were applied to setup calibration curves.
For every 10 samples, 1 blank sample was supplied. The limit of quantification and the limit of detection (LOD), as well as spiking recoveries, shown in

Egg health risk assessment
target hazard quotients (THQs) were calculated for the noncarcinogenic diseases by United States environmental protection agency (USEPA) method as the following equation (Means, 1989): When the THQ values are below one, the resident people will experience no potential health risk due to ingesting the product (Shaheen et al., 2016). Iranian daily egg consumption equals one egg/day/capita. However, in the current study, the own weight of each egg's white and yolk was used instead of the standard weight of an egg (60 g). The mean body weights of an adult and child (60 and 27 kg, respectively) were obtained from previous literature (Ki et al., 2017;Wang et al., 2005).

Statistical analysis
The sample size was calculated using a confidence level of 95%. The mean concentration of the metals was provided according to the mean frequency weights of the white and yolk. When the trace element concentration was below the instrument detection limit, mean LOD was inputted in the analysis (Ali et al., 2016). Pure data were entered into the regression analysis to take the regression coefficient of strain, feed, TA B L E 5 The number of mean concentrations of trace elements for feed in five types of poultry hens.

Effect of feed and strain on micronutrients concentration in egg
Cu: As presented in Tables 3 and 4, the highest concentration of Cu was found in the egg yolk of the LSL strain (2.074 mg/kg) and the lowest concentration in the egg white of the Hi-line strain (0.063 mg/kg). In the study of Bologa et al. (2014), the Cu values in total egg were 1.23-1.34 mg/kg wet weight, comparable to values found in this study. The mean Cu content of egg yolk was higher than that in the egg white for all the strains. The mean range of Cu content in the study of Salar-Amoli and Ali-Esfahani (2015) was 1.110-9.510 mg/kg, which was higher than the data obtained by this study. These discrepancies could be attributed to the different sampling procedures, sampling area and diet. The strain had a significant impact on egg Cu content. However, in the study of Salar-Amoli and Ali-Esfahani (2015), the effects of strains were negligible. Cu concentration in the diet ranged from 6.82 mg/kg in the LSL strain to 17.04 mg/kg in the Hi-line strain (Table 5) (Table 5).

None-metal Se:
Approximately 80% of ingested non-metal Se will be absorbed in the human gastrointestinal tract. The non-metal content of Se in foods depends on geographical locations, and it is necessary to monitor it regularly in soil and foods (Pinto et al., 2016). The none-metal Se had the lowest mean concentration after Co ranging from 0.038 mg/kg in the egg white of Hi-line strain to 0.763 mg/kg in the egg yolk of the native strain (Tables 3 and 4).
There were almost equal concentrations of non-metal Se in the white and yolk of the Bovans strain. Many studies reported a higher content of non-metal Se in yolk compared to the white in different strains (Dobrzański et al., 2007;Giannenas et al., 2009;Gjorgovska et al., 2012). There were significant differences in non-metal Se amounts only between Bovans and Native feed samples (Table 5). Giannenas et al. (2009) and Gjorgovska et al. (2012) reported a significant effect of dietary non-metal Se on egg non-metal Se deposition. Non-metal Se dietary content in Lohman LSL-Lite in the current study was 0.26 mg/kg, which is comparable with the reported nonmetal Se content (0.32 mg/kg) in the standard diet of Lohman Brown (Dobrzański et al., 2007).  (Table 5).
Therefore, it may be considered that the major source of Fe in the eggs is diet, as the former studies in the literature reported (Durge et al., 2022;Vincevica-Gaile et al., 2013). Fe content in the feed of selected strains ranged from 143.02 mg/kg in the native strain to 230.36 in the Bovans strain (Table 5).

TA B L E 6
The correlation of trace elements concentration in egg white, yolk and total eggs. Zn: Zn content had the second highest concentration in the eggs, and it was in the range of 0.346 mg/kg in egg white in Hi-line (Table 3) and 44.225 mg/kg in egg yolk in the native strain (Tables 4). Apart from Hi-line, the native strain had the highest Zn content in white (21.909 mg/kg) and yolk (44.225 mg/kg). As previously stated, the strain could have a significant effect on egg trace element content.

Part of egg
It seems that the effect of strain is more probable than diet in the case of Zn (Table 5). The data obtained by the current study (Tables 3   and 4) were inconsistent with the work of Dermilus et al., which reported 6.2 mg/kg in albumen and 43.9 mg/kg in the yolk, indicating that Zn values are higher in egg yolk than white (Demirulus, 2013). Bologa et al. (2014) found less Zn, ranging from 1.23 mg/kg wet weight to 1.49 mg/kg wet weight, which may be due to differences in the selected strain, the production systems (organic vs. conventional), or environmental factors such as the water and the feed.

Co:
The Co was not detected by the ICP-OES instrument (2 ppb, Table 2), especially in Shaver (Table 3) and Hi-line strains ( Table 4).
The lowest and the highest concentration of Co were found in the egg white of Hi-line (0.001 mg/kg fresh weight) and the egg yolk of LSL (0.023 mg/kg fresh weight), respectively. Chowdhury et al. (2011) revealed that Co concentration in samples of egg white was below the detection limit in farm hens, as well. Song et al. (2006) stated that the Co content ranged between 4.13 and 4.37 µg/kg in the egg samples. Moreover, a major difference in the Co content of eggs in different strains was observed (Tables 3 and 4). There was an association between eggs and diet for all the strains (Table 5). Similarly, Xiaoyong indicated the significant effect of the Co concentration of diet on the Co concentration of eggs (Lujiang et al., 1996). Tables 3 and 4  THQ is the ratio of the detected quantity of a pollutant to a standard reference dose level. The exposed population will likely suffer adverse effects if they expose to higher doses (Wang et al., 2005). This methodology does not provide a quantitative estimate of the probability of an exposed population experiencing a reverse health effect.

Mn: As stated in
But it shows a warning of the risk level due to contaminant exposure (Shaheen et al., 2016). Table 7 shows the mean values of THQs (dimensionless, THQs), standard deviations and p-values for Cu and Zn in Children and Adults according to different strains. Table 7 reveals that almost all the amount of THQs of Zn and Cu was less than even 0.1.
Therefore, the resident population was not experiencing the adverse effect caused by the consumption of eggs. Our results were in accordance with the study of Shaheen et al. (2016). In the case of children, regarding that they are more sensitive to harmful effects, this point is described in formulas by nearly half their weight, and more attention is needed. Although their THQs values were higher than that of adults, they were still in a safe range (Wang et al., 2005). There was a significant impact of strain on THQs in children and adults (p-value = 0.000, Table 7).

CONCLUSION
The current study assessed the effect of strain and diet on egg essential elements and surveyed the health risk of them. All the five essential elements had the lowest values in the Hi-line strain in both yolk and white and, consequently, in the whole egg. As stated in the Scheideler study, perhaps the Hi-line strain could have a more effective use of the diet. The potential health risk assessed by the THQs methodology was verified by US EPA, and their values were below one showing a safe range, but if all the trace elements take into account (sum of individual metal's THQs, the TTHQ), more than one TTHQ is probable. In addition, if the trace elements of the other major protein sources like meat and milk are considered too, probably the combined Hazardous Index (sum of TTHQs of different kinds of stuff) exceeds the acceptable limit, which may be a concerned authority on the potential health risks. All the amount of THQs of Zn and Cu was less than even 0.1. It is strongly recommended to evaluate the trace elements from food sources in the diet.